Surface modification of metals for biomolecule detection using surface enhanced Raman scattering (SERS)

ABSTRACT

The present invention is based on the discovery that the methods described herein for the production of metallic colloids result in colloids exhibiting increased signal enhancement and reproducibility for the SERS detection of biomolecules. Thus, using the methods of the invention, a wide variety of biomolecules can be detected with a greater sensitivity and reliability.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates generally to surface modified metallic particlesthat include organic molecules attached to the surface of metalliccolloids, and more specifically to the use of such surface modifiedparticles in analyte detection by surface-enhanced Raman spectroscopy.

2. Background Information

The ability to detect and identify trace quantities of analytes hasbecome increasingly important in virtually every scientific discipline,ranging from part per billion analyses of pollutants in sub-surfacewater to analysis of cancer treatment drugs in blood serum. Ramanspectroscopy is one analytical technique that provides richoptical-spectral information, and surface-enhanced Raman spectroscopy(SERS) has proven to be one of the most sensitive methods for performingquantitative and qualitative analyses. A Raman spectrum, similar to aninfrared spectrum, consists of a wavelength distribution of bandscorresponding to molecular vibrations specific to the sample beinganalyzed analyte). In the practice of Raman spectroscopy, the beam froma light source, generally a laser, is focused upon the sample to therebygenerate inelastically scattered radiation, which is optically collectedand directed into a wavelength-dispersive spectrometer in which adetector converts the energy of impinging photons to electrical signalintensity.

Among many analytical techniques that can be used for chemical structureanalysis, Raman spectroscopy is attractive for its capability inproviding rich structure information from a small optically-focused areaor detection cavity. Compared to a fluorescent spectrum that normallyhas a single peak with half peak width of tens of nanometers (quantumdots) to hundreds of nanometers (fluorescent dyes), a Raman spectrum hasmultiple bonding-structure-related peaks with half peak width of assmall as a few nanometers. Furthermore, surface enhanced Ramanscattering (SERS) techniques make it possible to obtain a 10⁶ to 10¹⁴fold Raman signal enhancement, and may even allow for single moleculedetection sensitivity. Such huge enhancement factors are attributedprimarily to enhanced electromagnetic fields on curved surfaces ofcoinage metals. Such enhancement factors have also been observed onsharp edges and at the junctions between aggregates. Although theelectromagnetic enhancement (EME) has been shown to be related to theroughness of metal surfaces or particle size when individual metalcolloids are used, SERS is most effectively detected from aggregatedcolloids. It is known that chemical enhancement can also be obtained byplacing molecules in a close proximity to the surface in certainorientations. Due to the rich spectral information and sensitivity,Raman signatures have been used as probe identifiers to detect a fewattomoles of molecules when SERS method was used to burst the signals ofspecifically immobilized Raman label molecules, which in fact are thedirect analytes of the SERS reaction. The method of attaching metalparticles to Raman-label-coated metal particles to obtain SERS-activecomplexes has also been studied. A recent study demonstrated that SERSsignal can be generated after attaching thiol containing dyes to goldparticle followed silica coating.

Unfortunately, reliable methods for producing metallic colloids withconsistent SERS performance have not yet been developed. In addition,there is a limited number of biomolecules (such as, for example,proteins) that adsorb to metallic surfaces to generate a SERS signal,and even for proteins that do adsorb, the signal intensity is low. Thus,a need exists for methods for producing metallic colloids withconsistent SERS performance for detection of biomolecules such asproteins. In addition, there exists a need for methods for producingmetallic colloids that are biomolecule specific.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a typical Surface Enhanced Raman Spectrum of dAMPobtained using the metallic colloids produced by the methods of theinvention.

FIG. 2 is a graph illustrating the reproducibility in SERS detection ofmetallic colloids produced by the methods of the invention.

FIG. 3 is a graphical depiction of a biomolecule being adsorbed ontoindividual metallic colloids produced by the methods of the inventionand aggregates thereof.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery that the methodsdescribed herein for the production of metal colloids result in colloidsexhibiting increased signal enhancement and reproducibility for the SERSdetection of biomolecules. Indeed, the surface modified metal colloidsprovided herein have the ability to adsorb specific target biomoleculesin a specific orientation. Indeed, the colloids employed in the methodsof the invention can be tailored for specific target biomolecules, forexample, certain colloids can be prepared for adsorbing proteins, whileother colloids can be prepared for adsorbing nucleic acids. Thus, usingthe methods of the invention, a wide variety of biomolecules can bedetected with a greater sensitivity and reliability.

In one embodiment, there are provided methods for producing metalliccolloids. Such methods can be performed, for example, by contactingmetal cations with a reducing agent in aqueous solution, and heating theaqueous solution to about 95° C., thereby producing metallic colloids.In other embodiments, the surface of the metallic colloids is modifiedby attaching an organic molecule to the surface of the colloids.

In another embodiment, there are provided methods for detecting abiomolecule in a sample. Such methods can be performed, for example, bymodifying a metallic surface with an organic molecule having affinitiesfor the metallic surface and for the biomolecule, contacting themodified metallic surface with the biomolecule, and detecting SERSsignals emitted by the biomolecule, wherein the signals are indicativeof the presence of the biomolecule.

In another embodiment, there are provided methods for detecting ananalyte in a sample. Such methods can be performed, for example, bycontacting a sample containing an analyte with a plurality of surfacemodified metallic colloids, wherein the analyte binds to the modifiedmetallic surface, and detecting SERS signals emitted by the analyte,wherein the signals are indicative of the presence of the analyte.

In another embodiment, there are provided systems for detecting ananalyte in a sample, including an array including more than one surfacemodified metallic colloid; a sample containing at least one analyte; aRaman spectrometer; and a computer including an algorithm for analysisof the sample.

In another embodiment, there are provided kits for labeling surfacemodified metallic colloids including a plurality of metallic colloidssurface modified according to the methods of the invention and abiological agent.

In another embodiment, there are provided methods of identifying amicroorganism. Such methods can be performed, for example, by contactinga sample suspected of containing the microorganism with an array ofmetallic colloids surface modified by the method of the invention;detecting SERS signals upon contacting the sample with the modifiedmetallic colloids and associating the SERS signals from the modifiedmetallic colloids with the identity of the microorganism.

In another embodiment, there are provided methods of identifying ananalyte. Such methods can be performed, for example, by contacting asample suspected of containing the analyte with an array of metalliccolloids surface modified by the method of the invention; detecting SERSsignals upon contacting the sample with the modified metallic colloids;and associating the SERS signals from the modified metallic colloidswith the identity of the analyte.

In one embodiment, there are provided methods for producing metalliccolloids. Such methods can be performed, for example, by contactingmetal cations with a reducing agent in aqueous solution, and heating theaqueous solution to about 95° C., thereby producing metallic colloids.As used herein, the term “colloid” refers to nanometer size metalparticles suspending in a liquid, usually an aqueous solution. In themethods of the invention, the metal cations and reducing agent are mixedin aqueous solution prior to heating. This method results in a 50%enhancement of SERS signals obtained from such colloids, and alsoresults in a increase in reproducibility of 10-20% to 80-100%. Typicalmetals contemplated for use in the practice of the invention include,for example, silver, gold, platinum, copper, aluminum, and the like.

A variety of reducing agents are contemplated for use in the practice ofthe invention, such as for example, citrate, borohydride, and the like.Sodium citrate is used in certain embodiments of the invention.Typically, the metal cation and reducing agent are each present inaqueous solution at a concentration of at least about 0.5 M. Aftermixing the metal cation and reducing agent, the solution is heated forabout 30 minutes. In some embodiments, the solution is heated for about60 minutes. Typically, the solution is heated to about 95° C. In otherembodiments, the solution is heated to about 100° C. Heating of thesolution is accomplished in a variety of ways well known to thoseskilled in the art. In some embodiments, the heating is accomplishedusing a microwave oven, a convection oven, or a combination thereof.

The methods for producing metallic colloids described herein are incontrast to prior methods wherein a boiling silver nitrate solution istitrated with a sodium citrate solution. This titration method canproduce only one batch of silver particles with adequate Ramanenhancement to dAMP in about 10 attempts, and the other batches have lowor no Raman activity at all. However, by employing the methods of theinvention, an average SERS signal enhancement of 150% is observedrelative to colloids prepared from the titration method.

In another embodiment of the invention, the metallic colloids producedby invention methods are modified by attaching an organic molecule tothe surface of the colloids. Organic molecules contemplated for use inthe practice of the invention are typically less than about 500 Daltonin molecular weight, and are bifunctional organic molecules. As usedherein, “bifunctional” means that the organic molecule has a moiety thathas an affinity for the metallic surface, and a moiety that has anaffinity for a biomolecule. Such surface modified metallic colloidsexhibit an increased ability to bind biomolecules, thereby resulting inan enhanced and reproducible SERS signal. The colloids can be usedeither individually, or as aggregates for binding certain biomolecules.

Organic molecules contemplated for use include molecules having anymoiety that exhibits an affinity for the metals contemplated for use inthe methods of the invention (i.e., silver, gold, platinum, copper,aluminum, and the like), and any moiety that exhibit affinities forbiomolecules. For example, with regard to silver or gold affinity, insome embodiments, the organic molecule has a sulfur containing moiety,such as for example, thiol, disulfide, and the like. With regard toaffinity for a biomolecule such as a polynucleotide, for example, theorganic molecule has a carboxylic acid moiety. In certain embodiments,the organic molecule is thiomalic acid, L-cysteine diethyl ester,S-carboxymethyl-L-cysteine, cystamine, meso-2,3-dimercaptosuccinic acid,and the like. It is understood, however, that any organic molecule thatmeets the definition of “bifunctional”, as described herein, iscontemplated for use in the practice of the invention. It is alsounderstood that the organic molecule may be attached to the metallicsurface and the biomolecule either covalently, or non-covalently.Indeed, the term “affinity” is intended to encompass the entire spectrumof chemical bonding interactions, which are well-known to those skilledin the art.

This surface modification of metallic colloids provides certainadvantages in SERS detection analyses. Since a wide variety of organicmolecules can be used in the invention methods, the surfaces of themetallic colloids can be tailored to bind to a specific biomolecule. Forexample, the surfaces can be tailored to differentiate among groups ofproteins based on the side chains of the individual amino acid residuesfound in the protein.

In other embodiments, there are provided methods for detecting abiomolecule in a sample. Such methods can be performed, for example, bymodifying a metallic surface with an organic molecule having affinitiesfor the metallic surface and for the biomolecule, contacting themodified metallic surface with the biomolecule, and detecting SERSsignals emitted by the biomolecule, wherein the signals are indicativeof the presence of the biomolecule.

The term “biomolecule” include antibodies, antigens, polynucleotides,oligonucleotides, receptors, ligands, and the like The term“polynucleotide” is used broadly herein to mean a sequence ofdeoxyribonucleotides or ribonucleotides that are linked together by aphosphodiester bond. For convenience, the term “oligonucleotide” is usedherein to refer to a polynucleotide that is used as a primer or a probe.Generally, an oligonucleotide useful as a probe or primer thatselectively hybridizes to a selected nucleotide sequence is at leastabout 10 nucleotides in length, usually at least about 15 nucleotides inlength, for example between about 15 and about 50 nucleotides in length.

A polynucleotide can be RNA or can be DNA, which can be a gene or aportion thereof, a cDNA, a synthetic polydeoxyribonucleic acid sequence,or the like, and can be single stranded or double stranded, as well as aDNA/RNA hybrid. In various embodiments, a polynucleotide, including anoligonucleotide (e.g., a probe or a primer) can contain nucleoside ornucleotide analogs, or a backbone bond other than a phosphodiester bond.In general, the nucleotides comprising a polynucleotide are naturallyoccurring deoxyribonucleotides, such as adenine, cytosine, guanine orthymine linked to 2′-deoxyribose, or ribonucleotides such as adenine,cytosine, guanine or uracil linked to ribose. However, a polynucleotideor oligonucleotide also can contain nucleotide analogs, includingnon-naturally occurring synthetic nucleotides or modified naturallyoccurring nucleotides.

The covalent bond linking the nucleotides of a polynucleotide generallyis a phosphodiester bond. However, the covalent bond also can be any ofnumerous other bonds, including a thiodiester bond, a phosphorothioatebond, a peptide-like amide bond or any other bond known to those in theart as useful for linking nucleotides to produce syntheticpolynucleotides. The incorporation of non-naturally occurring nucleotideanalogs or bonds linking the nucleotides or analogs can be particularlyuseful where the polynucleotide is to be exposed to an environment thatcan contain a nucleolytic activity, including, for example, a tissueculture medium or upon administration to a living subject, since themodified polynucleotides can be less susceptible to degradation.

As used herein, the term “selective hybridization” or “selectivelyhybridize,” refers to hybridization under moderately stringent or highlystringent conditions such that a nucleotide sequence preferentiallyassociates with a selected nucleotide sequence over unrelated nucleotidesequences to a large enough extent to be useful in identifying theselected nucleotide sequence. It will be recognized that some amount ofnon-specific hybridization is unavoidable, but is acceptable providedthat hybridization to a target nucleotide sequence is sufficientlyselective such that it can be distinguished over the non-specificcross-hybridization, for example, at least about 2-fold more selective,generally at least about 3-fold more selective, usually at least about5-fold more selective, and particularly at least about 10-fold moreselective, as determined, for example, by an amount of labeledoligonucleotide that binds to target nucleic acid molecule as comparedto a nucleic acid molecule other than the target molecule, particularlya substantially similar (i.e., homologous) nucleic acid molecule otherthan the target nucleic acid molecule. Conditions that allow forselective hybridization can be determined empirically, or can beestimated based, for example, on the relative GC:AT content of thehybridizing oligonucleotide and the sequence to which it is tohybridize, the length of the hybridizing oligonucleotide, and thenumber, if any, of mismatches between the oligonucleotide and sequenceto which it is to hybridize.

An example of progressively higher stringency conditions is as follows:2×SSC/0.1% SDS at about room temperature (hybridization conditions);0.2×SSC/0.1% SDS at about room temperature (low stringency conditions);0.2×SSC/0.1% SDS at about 42EC (moderate stringency conditions); and0.1×SSC at about 68EC (high stringency conditions). Washing can becarried out using only one of these conditions, e.g., high stringencyconditions, or each of the conditions can be used, e.g., for 10-15minutes each, in the order listed above, repeating any or all of thesteps listed. However, as mentioned above, optimal conditions will vary,depending on the particular hybridization reaction involved, and can bedetermined empirically.

In some embodiments, the biomolecule is an antibody. As used herein, theterm “antibody” is used in its broadest sense to include polyclonal andmonoclonal antibodies, as well as antigen binding fragments of suchantibodies. An antibody useful in a method of the invention, or anantigen binding fragment thereof, is characterized, for example, byhaving specific binding activity for an epitope of an analyte.

The antibody, for example, includes naturally occurring antibodies aswell as non-naturally occurring antibodies, including, for example,single chain antibodies, chimeric, bifunctional and humanizedantibodies, as well as antigen-binding fragments thereof. Suchnon-naturally occurring antibodies can be constructed using solid phasepeptide synthesis, can be produced recombinantly or can be obtained, forexample, by screening combinatorial libraries consisting of variableheavy chains and variable light chains. These and other methods ofmaking, for example, chimeric, humanized, CDR-grafted, single chain, andbifunctional antibodies are well known to those skilled in the art.

The term “binds specifically” or “specific binding activity,” when usedin reference to an antibody means that an interaction of the antibodyand a particular epitope has a dissociation constant of at least about1×10⁻⁶, generally at least about 1×10⁻⁷, usually at least about 1×10⁻⁸,and particularly at least about 1×10⁻⁹ or 1×10⁻¹⁰or less. As such, Fab,F(ab′)₂, Fd and Fv fragments of an antibody that retain specific bindingactivity for an epitope of an antigen, are included within thedefinition of an antibody.

In the context of the invention, the term “ligand” denotes a naturallyoccurring specific binding partner of a receptor, a syntheticspecific-binding partner of a receptor, or an appropriate derivative ofthe natural or synthetic ligands. As one of skill in the art willrecognize, a biomolecule (or macromolecular complex) can be both areceptor and a ligand. In general, the binding partner having a smallermolecular weight is referred to as the ligand and the binding partnerhaving a greater molecular weight is referred to as a receptor.

In other embodiments, there are provided methods for detecting ananalyte in a sample. Such methods can be performed for example, bycontacting a sample containing an analyte with a plurality of surfacemodified metallic colloids, wherein the analyte binds to the modifiedmetallic surface, and detecting SERS signals emitted by the analyte,wherein the signals are indicative of the presence of the analyte. Inthis embodiment, the surface of the metallic colloid is modified byattaching an organic molecule to the surface of the colloid, wherein theorganic molecule has an affinity for both the metallic surface and theanalyte.

By “analyte” is meant any molecule or compound. An analyte can be in thesolid, liquid, gaseous or vapor phase. By “gaseous or vapor phaseanalyte” is meant a molecule or compound that is present, for example,in the headspace of a liquid, in ambient air, in a breath sample, in agas, or as a contaminant in any of the foregoing. It will be recognizedthat the physical state of the gas or vapor phase can be changed bypressure, temperature as well as by affecting surface tension of aliquid by the presence of or addition of salts etc.

The analyte can be comprised of a member of a specific binding pair(sbp) and may be a ligand, which is monovalent (monoepitopic) orpolyvalent (polyepitopic), usually antigenic or haptenic, and is asingle compound or plurality of compounds which share at least onecommon epitopic or determinant site. The analyte can be a part of a cellsuch as bacteria or a cell bearing a blood group antigen such as A, B,D, etc., or an HLA antigen or a microorganism, e.g., bacterium, fungus,protozoan, or virus. In certain aspects of the invention, the analyte ischarged.

A member of a specific binding pair (“sbp member”) is one of twodifferent molecules, having an area on the surface or in a cavity whichspecifically binds to and is thereby defined as complementary with aparticular spatial and polar organization of the other molecule. Themembers of the specific binding pair are referred to as ligand andreceptor (antiligand) or analyte and probe. Therefore, a probe is amolecule that specifically binds an analyte. These will usually bemembers of an immunological pair such as antigen-antibody, althoughother specific binding pairs such as biotin-avidin, hormones-hormonereceptors, nucleic acid duplexes, IgG-protein A, polynucleotide pairssuch as DNA-DNA, DNA-RNA, and the like are not immunological pairs butare included in the invention and the definition of sbp member.

Specific binding is the specific recognition of one of two differentmolecules for the other compared to substantially less recognition ofother molecules. Generally, the molecules have areas on their surfacesor in cavities giving rise to specific recognition between the twomolecules. Exemplary of specific binding are antibody-antigeninteractions, enzyme—substrate interactions, polynucleotidehybridization interactions, and so forth.

Non-specific binding is non-covalent binding between molecules that isrelatively independent of specific surface structures. Non-specificbinding may result from several factors including hydrophobicinteractions between molecules.

The methods of the present invention may be used to detect the presenceof a particular target analyte, for example, a nucleic acid,oligonucleotide, protein, enzyme, antibody or antigen. The methods mayalso be used to screen bioactive agents, i.e. drug candidates, forbinding to a particular target or to detect agents like pollutants.

The polyvalent ligand analytes will normally be poly(amino acids), i.e.,polypeptides and proteins, polysaccharides, nucleic acids, andcombinations thereof. Such combinations include components of bacteria,viruses, chromosomes, genes, mitochondria, nuclei, cell membranes andthe like.

For the most part, the polyepitopic ligand analytes to which the subjectinvention can be applied will have a molecular weight of at least about5,000, more usually at least about 10,000. In the poly(amino acid)category, the poly(amino acids) of interest will generally be from about5,000 to 5,000,000 molecular weight, more usually from about 20,000 to1,000,000 molecular weight; among the hormones of interest, themolecular weights will usually range from about 5,000 to 60,000molecular weight.

The monoepitopic ligand analytes will generally be from about 100 to2,000 molecular weight, more usually from 125 to 1,000 molecular weight.The analytes include drugs, metabolites, pesticides, pollutants, and thelike. Included among drugs of interest are the alkaloids. Among thealkaloids are morphine alkaloids, which includes morphine, codeine,heroin, dextromethorphan, their derivatives and metabolites; cocainealkaloids, which include cocaine and benzyl ecgonine, their derivativesand metabolites; ergot alkaloids, which include the diethylamide oflysergic acid; steroid alkaloids; iminazoyl alkaloids; quinazolinealkaloids; isoquinoline alkaloids; quinoline alkaloids, which includequinine and quinidine; diterpene alkaloids, their derivatives andmetabolites.

The term analyte further includes polynucleotide analytes such as thosepolynucleotides defined below. These include m-RNA, r-RNA, t-RNA, DNA,DNA-RNA duplexes, etc. The term analyte also includes receptors that arepolynucleotide binding agents, such as, for example, peptide nucleicacids (PNA), restriction enzymes, activators, repressors, nucleases,polymerases, histones, repair enzymes, chemotherapeutic agents, and thelike.

The analyte may be a molecule found directly in a sample such as a bodyfluid from a host. The sample can be examined directly or may bepretreated to render the analyte more readily detectible. Furthermore,the analyte of interest may be determined by detecting an agentprobative of the analyte of interest such as a specific binding pairmember complementary to the analyte of interest, whose presence will bedetected only when the analyte of interest is present in a sample. Thus,the agent probative of the analyte becomes the analyte that is detectedin an assay. The body fluid can be, for example, urine, blood, plasma,serum, saliva, semen, stool, sputum, cerebral spinal fluid, tears,mucus, and the like.

In the methods of the invention, a “sample” includes a wide variety ofanalytes that can be analyzed using the methods described herein, solong as the subject analyte is capable of generating SERS signals uponlaser irradiation. For example, a sample can be an environmental sampleand includes atmospheric air, ambient air, water, sludge, soil, and thelike. In addition, a sample can be a biological sample, including, forexample, a subject's breath, saliva, blood, urine, feces, varioustissues, and the like.

Commercial applications for the invention methods employing the methodsdescribed herein include environmental toxicology and remediation,biomedicine, materials quality control, food and agricultural productsmonitoring, anaesthetic detection, automobile oil or radiator fluidmonitoring, breath alcohol analyzers, hazardous spill identification,explosives detection, fugitive emission identification, medicaldiagnostics, fish freshness, detection and classification of bacteriaand microorganisms both in vitro and in vivo for biomedical uses andmedical diagnostic uses, monitoring heavy industrial manufacturing,ambient air monitoring, worker protection, emissions control, productquality testing, leak detection and identification, oil/gaspetrochemical applications, combustible gas detection, H₂S monitoring,hazardous leak detection and identification, emergency response and lawenforcement applications, illegal substance detection andidentification, arson investigation, enclosed space surveying, utilityand power applications, emissions monitoring, transformer faultdetection, food/beverage/agriculture applications, freshness detection,fruit ripening control, fermentation process monitoring and controlapplications, flavor composition and identification, product quality andidentification, refrigerant and fumigant detection,cosmetic/perfume/fragrance formulation, product quality testing,personal identification, chemical/plastics/pharmaceutical applications,leak detection, solvent recovery effectiveness, perimeter monitoring,product quality testing, hazardous waste site applications, fugitiveemission detection and identification, leak detection andidentification, perimeter monitoring, transportation, hazardous spillmonitoring, refueling operations, shipping container inspection,diesel/gasoline/aviation fuel identification, building/residentialnatural gas detection, formaldehyde detection, smoke detection, firedetection, automatic ventilation control applications (cooking, smoking,etc.), air intake monitoring, hospital/medical anesthesia &sterilization gas detection, infectious disease detection and breathapplications, body fluids analysis, pharmaceutical applications, drugdiscovery, telesurgery, and the like.

Another application for the sensor-based fluid detection device inengine fluids is an oil/antifreeze monitor, engine diagnostics forair/fuel optimization, diesel fuel quality, volatile organic carbonmeasurement (VOC), fugitive gases in refineries, food quality,halitosis, soil and water contaminants, air quality monitoring, leakdetection, fire safety, chemical weapons identification, use byhazardous material teams, explosive detection, breathalyzers, ethyleneoxide detectors and anaesthetics.

In another embodiment, there are provided systems for detecting ananalyte in a sample. Such systems include, an array comprising more thanone surface modified metallic colloid; a sample containing at least oneanalyte; a Raman spectrometer; and a computer including an algorithm foranalysis of the sample.

A variety of analytical techniques can be used to analyze the samplesdescribed herein. Such techniques include for example, nuclear magneticresonance spectroscopy (NMR), photon correlation spectroscopy (PCS), IR,surface plasma resonance (SPR), XPS, scanning probe microscopy (SPM),SEM, TEM, atomic absorption spectroscopy, elemental analysis, UV-vis,fluorescence spectroscopy, and the like.

In the practice of the present invention, the Raman spectrometer can bepart of a detection unit designed to detect and quantify metalliccolloids of the present invention by Raman spectroscopy. Methods fordetection of Raman labeled analytes, for example nucleotides, usingRaman spectroscopy are known in the art. (See, e.g., U.S. Pat. Nos.5,306,403; 6,002,471; 6,174,677). Variations on surface enhanced Ramanspectroscopy (SERS), surface enhanced resonance Raman spectroscopy(SERRS) and coherent anti-Stokes Raman spectroscopy (CARS) have beendisclosed.

A non-limiting example of a Raman detection unit is disclosed in U.S.Pat. No. 6,002,471. An excitation beam is generated by either afrequency doubled Nd:YAG laser at 532 nm wavelength or a frequencydoubled Ti:sapphire laser at 365 nm wavelength. Pulsed laser beams orcontinuous laser beams may be used. The excitation beam passes throughconfocal optics and a microscope objective, and is focused onto the flowpath and/or the flow-through cell. The Raman emission light from thelabeled silver colloids is collected by the microscope objective and theconfocal optics and is coupled to a monochromator for spectraldissociation. The confocal optics includes a combination of dichroicfilters, barrier filters, confocal pinholes, lenses, and mirrors forreducing the background signal. Standard full field optics can be usedas well as confocal optics. The Raman emission signal is detected by aRaman detector, that includes an avalanche photodiode interfaced with acomputer for counting and digitization of the signal.

Another example of a Raman detection unit is disclosed in U.S. Pat. No.5,306,403, including a Spex Model 1403 double-grating spectrophotometerwith a gallium-arsenide photomultiplier tube (RCA Model C31034 or BurleIndustries Model C3103402) operated in the single-photon counting mode.The excitation source includes a 514.5 nm line argon-ion laser fromSpectraPhysics, Model 166, and a 647.1 nm line of a krypton-ion laser(Innova 70, Coherent).

Alternative excitation sources include a nitrogen laser (Laser ScienceInc.) at 337 nm and a helium-cadmium laser (Liconox) at 325 nm (U.S.Pat. No. 6,174,677), a light emitting diode, an Nd:YLF laser, and/orvarious ions lasers and/or dye lasers. The excitation beam may bespectrally purified with a bandpass filter (Corion) and may be focusedon the flow path and/or flow-through cell using a 6× objective lens(Newport, Model L6X). The objective lens may be used to both excite theanalyte and to collect the Raman signal, by using a holographic beamsplitter (Kaiser Optical Systems, Inc., Model HB 647-26N18) to produce aright-angle geometry for the excitation beam and the emitted Ramansignal. A holographic notch filter (Kaiser Optical Systems, Inc.) may beused to reduce Rayleigh scattered radiation. Alternative Raman detectorsinclude an ISA HR-320 spectrograph equipped with a red-enhancedintensified charge-coupled device (RE-ICCD) detection system (PrincetonInstruments). Other types of detectors may be used, such asFourier-transform spectrographs (based on Michaelson interferometers),charged injection devices, photodiode arrays, InGaAs detectors,electron-multiplied CCD, intensified CCD and/or phototransistor arrays.

Any suitable form or configuration of Raman spectroscopy or relatedtechniques known in the art may be used for detection in the methods ofthe present invention, including but not limited to normal Ramanscattering, resonance Raman scattering, surface enhanced Ramanscattering, surface enhanced resonance Raman scattering, coherentanti-Stokes Raman spectroscopy (CARS), stimulated Raman scattering,inverse Raman spectroscopy, stimulated gain Raman spectroscopy,hyper-Raman scattering, molecular optical laser examiner (MOLE) or Ramanmicroprobe or Raman microscopy or confocal Raman microspectrometry,three-dimensional or scanning Raman, Raman saturation spectroscopy, timeresolved resonance Raman, Raman decoupling spectroscopy or UV-Ramanmicroscopy.

In certain aspects of the invention, a system for detecting an analyteof the present invention includes an information processing system. Anexemplary information processing system may incorporate a computer thatincludes a bus for communicating information and a processor forprocessing information. In one embodiment of the invention, theprocessor is selected from the Pentium® family of processors, includingwithout limitation the Pentium® II family, the Pentium® III family andthe Pentium® 4 family of processors available from Intel Corp. (SantaClara, Calif.). In alternative embodiments of the invention, theprocessor may be a Celeron®, an Itanium®, or a Pentium Xeon® processor(Intel Corp., Santa Clara, Calif.). In various other embodiments of theinvention, the processor may be based on Intel® architecture, such asIntel® IA-32 or Intel® IA-64 architecture. Alternatively, otherprocessors may be used. The information processing and control systemmay further comprise any peripheral devices known in the art, such asmemory, display, keyboard and/or other devices.

In particular examples, the detection unit can be operably coupled tothe information processing system. Data from the detection unit may beprocessed by the processor and data stored in memory. Data on emissionprofiles for various raman labels may also be stored in memory. Theprocessor may compare the emission spectra from the sample in the flowpath and/or flow-through cell to identify the raman-active organiccompound. The processor may analyze the data from the detection unit todetermine, for example, the sequence of a polynucleotide bound by asilver colloid employed by the methods of the present invention. Theinformation processing system may also perform standard procedures suchas subtraction of background signals

While certain methods of the present invention may be performed underthe control of a programmed processor, in alternative embodiments of theinvention, the methods may be fully or partially implemented by anyprogrammable or hardcoded logic, such as Field Programmable Gate Arrays(FPGAs), TTL logic, or Application Specific Integrated Circuits (ASICs).Additionally, the disclosed methods may be performed by any combinationof programmed general purpose computer components and/or custom hardwarecomponents.

Following the data gathering operation, the data will typically bereported to a data analysis operation. To facilitate the analysisoperation, the data obtained by the detection unit will typically beanalyzed using a digital computer such as that described above.Typically, the computer will be appropriately programmed for receipt andstorage of the data from the detection unit as well as for analysis andreporting of the data gathered.

In certain embodiments of the invention, custom designed softwarepackages may be used to analyze the data obtained from the detectionunit. In alternative embodiments of the invention, data analysis may beperformed, using an information processing system and publicly availablesoftware packages.

The invention will be further understood with reference to the followingexamples, which are purely exemplary, and should not be taken aslimiting the true scope of the present invention as described in theclaims.

EXAMPLE 1 Preparation of Silver Colloids

A: To a 250 mL round bottom flask equipped with a stirring bar, wasadded 100 mL de-ionized water and 0.200 mL of a 0.500 M silver nitratesolution. The flask was shaken to thoroughly mix the solution. 0.136 mLof a 0.500 M sodium citrate solution was then added to the flask using a200 μl pipette. The flask was then placed in a heating mantle and thestirrer was set at medium speed. A water cooled condenser was attachedto the flask and heating commenced. The heating mantle was applied atmaximum voltage, resulting in boiling of the solution between 7 and 10minutes. Color changes occur within 120 seconds of boiling. The heatingis stopped after 60 minutes, the solution is cooled to room temperatureand the resulting colloidal suspension is transferred to a 100 mL glassbottle for storage.

B: To a 250 mL Pyrex glass bottle with 100 mL ultrapure water, wasadded 1. mL of a 0.500M silver nitrate solution. This solution was mixedwell prior to addition of 0.5 mL of a 0.500M sodium citrate solution.About 3 grams of PTFE boiling stones (6 mm, VWR) were then placed intothe bottle. A cap was placed on the bottle but not screwed tightly, andthe bottle was placed in the center of a microwave oven (1350 Watt,Panasonic Model NN_S553BF, Type S333). The heating profile wasprogrammed as follows: P10 (maximum power) for 90 seconds and P2 (20%maximum power) for 3:30 minutes. The solution begins to boil in about 60seconds and color changes occur at 110-120 seconds. After heating themicrowave for 5 minutes, the bottle is immediately removed andtransferred to a convection oven set at 95° C. (model MO1440SA,Lindberg/Blue, Ashville, N.C.). The bottle is removed from theconvection oven and the final volume of the suspension is adjusted to100 mL with ultrapure water. A typical Raman spectrum is given inFIG. 1. To compare the Raman enhancement with the best lot obtained froma titration method, the peak height at wave number 1320 cm⁻¹ wasnormalized by that of the best lot from the titration method. Data fromseveral preparations using the microwave method are given in FIG. 2.

Although the invention has been described with reference to the aboveexample, it will be understood that modifications and variations areencompassed within the spirit and scope of the invention. Accordingly,the invention is limited only by the following claims.

1. A method for producing a cluster of surface-modified metallic colloidcomprising (1) metallic colloid comprising a metal and (2) aRaman-enhancing organic molecule on a surface of the metallic colloid,the method comprising: preparing a solution comprising cations of themetal and a reducing agent by dissolving the cations and the reducingagent in the solution, subsequently heating the solution to produce themetallic colloid, modifying the metallic colloid by attaching the Ramanenhancing organic molecule to the surface of the metallic colloid toproduce the surface modified metallic colloid, wherein theRaman-enhancing organic molecule comprises a moiety that has an affinityfor the metallic colloid and another moiety that has an affinity for abiomolecule, and aggregating a plurality of the surface-modifiedmetallic colloid to form the cluster of surface-modified metalliccolloid.
 2. The method of claim 1, wherein the reducing agent is citrateor borohydride.
 3. The method of claim 1, wherein said heating isperformed for at least about 30 minutes.
 4. The method of claim 1,wherein said heating is performed for at least about 60 minutes.
 5. Themethod of claim 1, wherein said heating is performed using microwaves.6. The method of claim 1, wherein said heating is performed using aconvection oven.
 7. The method of claim 1, wherein the metal is silver,gold, platinum, or aluminum.
 8. The method of claim 1, wherein theRaman-enhancing organic molecule is a bifunctional organic molecule. 9.The method of claim 1, wherein the Raman-enhancing organic moleculecontains sulfur.
 10. The method of claim 1, wherein the Raman-enhancingorganic molecule has a molecular weight less than about 500 Daltons. 11.The method of claim 1, wherein the Raman-enhancing organic moleculecontains a thiol moiety or a disulfide moiety.
 12. The method of claim1, wherein the Raman-enhancing organic molecule is thiomalic acid,L-cysteine diethyl ester, S-carboxymethyl-L-cysteine, cystamine, ormeso-2,3-dimercaptosuccinic acid.
 13. The method of claim 1, wherein thesolution is an aqueous solution.
 14. The method of claim 1, wherein saidsubsequent heating the solution is performed at a temperature of about95° C.
 15. The method of claim 1, wherein the metallic colloid producesa higher SERS signal than that produced by another metallic colloidcomprising the same metal except that the another metallic colloid isproduced by titrating the cations and the reducing agent in the solutionat a near boiling point temperature.
 16. The method of claim 15, whereinthe metallic colloid produces at least about 50% higher SERS signal thanthat produced by the another metallic colloid.
 17. The method of claim1, wherein the cations and reducing agent are each present in theaqueous solution at a concentration of about 0.5 M or higher than 0.5 M.18. The method of claim 1, wherein the metallic colloid has a Ramansignal that is 50% or more than that of a silver colloid prepared by atitration method wherein a boiling silver nitrate solution is titratedwith a sodium citrate solution to produce the silver colloid.
 19. Themethod of claim 1, wherein the metallic colloid is formed by aggregatinga plurality of the metallic particles to form clusters ranging fromabout 50 nm to 200 nm.
 20. A method for producing a cluster ofsurface-modified metallic colloid comprising (1) metallic colloidcomprising a metal and (2) a Raman-enhancing organic molecule on asurface of the metallic colloid, the method comprising: preparing asolution comprising cations of the metal and a reducing agent bydissolving the cations and the reducing agent in the solution,subsequently heating the solution to produce the metallic colloid,modifying the metallic colloid by attaching the Raman-enhancing organicmolecule to the surface of the metallic colloid to produce thesurface-modified metallic colloid, wherein the Raman-enhancing organicmolecule comprises a moiety that has an affinity for the metalliccolloid and another moiety that has an affinity for a biomolecule, andaggregating a plurality of the surface-modified metallic colloid to formthe cluster of surface-modified metallic colloid. wherein the clusterhas a size ranging from about 50 nm to 200 nm.
 21. The method of claim1, wherein the metallic colloid has a Raman signal that is about 140% to180% more than that of a silver colloid prepared by a titration methodwherein a boiling silver nitrate solution is titrated with a sodiumcitrate solution to produce the silver colloid.
 22. The method of claim1, wherein the cluster of surface-modified metallic colloid has a Ramansignal that is greater than that of a silver colloid prepared by atitration method wherein a boiling silver nitrate solution is titratedwith a sodium citrate solution to produce the silver colloid.